Endothelial cells that form the lining of blood vessels are well known for their capacity to adjust their numbers and arrangement to suite local requirements. All tissues depend on a blood supply and the blood supply depends on endothelial cells. Blood vessels create an adaptable life support system in every region of the body. If not for endothelial cells extending and maintaining this network of blood vessels, tissue growth and repair would not be possible.
The largest blood vessels are arteries and veins, which have a thick tough outer wall of connective tissue and smooth muscle. The wall is lined by a thin single layer of endothelial cells, separated from the surrounding outer layers by a basal lamina. While the amounts of connective-tissue and smooth muscle in the vessel wall may vary according to the vessel's diameter and function, the endothelial lining is always present. In the smaller capillaries and sinusoids, the walls consist solely of endothelial cells and basal lamina Thus, endothelial cells line the entire vascular system. Studies have shown that arteries and veins develop from small vessels constructed solely of endothelial cells and a basal lamina, connective tissue and smooth muscle being added later where required upon signals from the endothelial cells.
Throughout the vascular system endothelial cells retain a capacity for cell division and movement. This is important in repair and maintenance of the vascular system. For example, if a part of the wall of a blood vessel is damaged and loses endothelial cells, neighboring endothelial cells will proliferate and migrate in to cover the exposed surface. Newly formed endothelial cells have also been known to cover the inner surface of plastic tubing used by surgeons to replace damaged blood vessels.
Endothelial cells not only repair damaged blood vessels, they also create new blood vessels. Endothelial cells do this in embryonic tissues to support growth, in normal adult tissue for repair and maintenance, and in damaged tissue to support repair. This process is called angiogenesis.
Angiogenesis is a critical component in embryonic development, tissue growth, tissue remodeling, and a number of pathologies. Angiogenesis results in the formation of new blood vessels. During angiogenesis, endothelial cells, which exist in a quiescent state as part of an existing blood vessel, grow and enter a migratory, proliferative state. This migratory, proliferative state is eventually resolved when the cells differentiate into capillary tubes and return to the quiescent state as part of a functional new blood vessel. Angiogenesis is orchestrated by a complex network of multiple macromolecular interactions.
Angiogenesis is regulated in both normal and malignant tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites. Vascular endothelial growth factor-A (VEGF, also known as vascular permeability factor, VPF) is a primary stimulant of angiogenesis. VEGF is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations and can be produced by a wide variety of tissues.
Angiogenesis is stimulated and harnessed by some neoplasms (e.g., tumors) to increase nutrient uptake. However, in contrast to normal angiogenesis, which leads to anastomoses (i.e., vessel connections) and capillary maturation, angiogenesis associated with neoplasia is typically a continuous process where vessel maturation is imperfect. Endothelial cells are activated by nearby neoplastic cells to secrete not only VEGF which stimulates angiogenesis, but also matrix metalloproteases (MMP) which degrade the surrounding extracellular matrix. The endothelial cells then invade the extracellular matrix where they proliferate, migrate, and organize to form new blood vessels, which support neoplasm growth and survival.
The newly vascularized neoplasm continues to grow, leading to further nutrient deprivation and chronic pro-angiogenic signaling. The vasculature of neoplasms is characterized by the presence of structural irregularities (lacunae) and a low rate of formation of inter-vessel connections. This incomplete vasculature is inefficient, such that often tumors require continuous angiogenesis to sustain themselves. Such imperfect vasculature is also believed to promote the shedding of neoplastic cells into the systemic circulation. Hence, the angiogenic potential of a neoplasm correlates with metastatic potential. As a significant proportion of neoplasms are dependent on continued angiogenesis, inhibition of angiogenesis blocks neoplasm growth which often leads to complete necrosis of the neoplasm.
Glial cells including astrocytes comprise a large proportion of the total cell population in the central nervous system. Unlike neurons, glial cells retain the ability to proliferate postnatally, and some glial cells still proliferate in the adult or aged brain. Uncontrolled glial proliferation can lead to aggressive primary intracranial tumors. Such tumors vary widely in morphology and behavior, and, according to the 1993 World Health Organization classification schema, can be separated into three subsets. Astrocytomas, the lowest grade tumors, are generally well-differentiated and tend to grow slowly. Anaplastic astrocytomas are characterized by increased cellularity, nuclear pleomorphism (ability to assume different forms), and increased mitotic activity. They are intermediate grade tumors and show a tendency to progress to a more aggressive grade. Glioblastoma cells are considered the most aggressive, with poorly differentiated cells, vascular proliferation, and necrosis. Glioblastoma U251 is a malignant cell line derived from the human glial cells.
The angiogenic effects of glioblastoma cells and other solid tumor cells in the presence of in vivo matrix effects and other in vivo ancillary factors, do not predict in vitro effectiveness in inducing primary endothelial cells in culture to grow, and more particularly do not predict in vitro effectiveness in inducing primary endothelial cells in culture to form tubules. The effectiveness of very low numbers of tumor cells to induce this effect is still more unexpected.
Non-normal, i.e., immortalized endothelial cells have been reported to form tubular structures in culture in the presence of glioblastoma cells. But the growth of these kinds of cells can be expected to differ considerably from normal cells, so the induction of tubules in normal cells could not be reliably predicted from this earlier work.
Thus, there is a need to induce angiogenic endothelial cells to better enable collection of angiogenic endothelial cells for such purposes as angiogenic assay kits and in the study of endothelial cells, particularly the functions and permeability of the endothelial cell barrier. Further, such cells have potential therapeutic uses.